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Excellent short-term survival after pediatric liver transplantation (LT) has shifted attention toward the optimization of long-term outcomes. Despite considerable progress in imaging and other noninvasive modalities, liver biopsies continue to be required to monitor allograft health and to titrate immunosuppression. However, a standardized approach to the detailed assessment of long-term graft histology is currently lacking. The aim of this study was to formulate a list of histopathological features relevant for the assessment of long-surviving liver allograft health and to develop an approach for assessing the presence and severity of these features in a standardized manner. Whole-slide digital images from 31 biopsies obtained ≥4 years after transplantation to determine eligibility for an immunosuppression withdrawal trial were selected to illustrate a range of typical histopathological findings seen in children with clinically stable grafts, including those associated with alloantibodies. Fifty histological features were independently assessed and, where appropriate, scored semiquantitatively by six pathologists to determine inter- and intraobserver reproducibility of the histopathological features using unweighted and weighted kappa statistics; the latter metric enabled distinction between minor and major disagreements in parameter severity scoring. Weighted interobserver kappa statistics showed a high level of agreement for various parameters of inflammation, interface activity, fibrosis, and microvascular injury. Intraobserver agreement for these features was even more substantial. The results of this study will help to standardize the assessment of biopsies from long-surviving liver allografts, aid the recognition of important histological features, and facilitate international comparisons and clinical trials aiming to improve outcomes for children undergoing LT., (© 2022 American Association for the Study of Liver Diseases.)

Background and Aims: Nonanastomotic biliary strictures (NAS) are a major cause of morbidity after orthotopic liver transplantation (OLT). Although ischemic injury of peribiliary glands (PBGs) and peribiliary vascular plexus during OLT has been associated with the later development of NAS, the exact underlying mechanisms remain unclear. We hypothesized that bile ducts of patients with NAS suffer from ongoing biliary hypoxia and lack of regeneration from PBG stem/progenitor cells., Approach and Results: Forty-two patients, requiring retransplantation for either NAS (n = 18), hepatic artery thrombosis (HAT; n = 13), or nonbiliary graft failure (controls; n = 11), were included in this study. Histomorphological analysis of perihilar bile ducts was performed to assess differences in markers of cell proliferation and differentiation in PBGs, microvascular density (MVD), and hypoxia. In addition, isolated human biliary tree stem cells (hBTSCs) were used to examine exo-metabolomics during in vitro differentiation toward mature cholangiocytes. Bile ducts of patients with NAS or HAT had significantly reduced indices of PBG mass, cellular proliferation and differentiation (mucus production, secretin receptor expression, and primary cilia), reduced MVD, and increased PBG apoptosis and hypoxia marker expression, compared to controls. Metabolomics of hBTSCs during in vitro differentiation toward cholangiocytes revealed a switch from a glycolytic to oxidative metabolism, indicating the need for oxygen., Conclusions: NAS are characterized by a microscopic phenotype of chronic biliary hypoxia attributed to loss of microvasculature, resulting in reduced proliferation and differentiation of PBG stem/progenitor cells into mature cholangiocytes. These findings suggest that persistent biliary hypoxia is a key mechanism underlying the development of NAS after OLT., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background: Ex situ normothermic machine perfusion (NMP) can be used to assess viability of suboptimal donor livers before implantation. Our aim was to assess the diagnostic accuracy of bile biochemistry for the assessment of bile duct injury (BDI)., Methods: In a preclinical study, 23 human donor livers underwent 6 hours of end-ischemic NMP to determine biomarkers of BDI. Livers were divided into groups with low or high BDI, based on a clinically relevant histological grading system. During NMP, bile was analyzed biochemically and potential biomarkers were correlated with the degree of BDI. Receiver operating characteristics curves were generated to determine optimal cutoff values. For clinical validation, identified biomarkers were subsequently included as viability criteria in a clinical trial (n = 6) to identify transplantable liver grafts with low BDI., Results: Biliary bicarbonate and pH were significantly higher and biliary glucose was significantly lower in livers with low BDI, compared with high BDI. The following cutoff values were associated with low BDI: biliary bicarbonate greater than 18 mmol/L (P = 0.002), biliary pH greater than 7.48 (P = 0.019), biliary glucose less than 16 mmol/L (P = 0.013), and bile/perfusate glucose ratio less than 0.67 (P = 0.013). In the clinical trial, 4 of 6 livers met these criteria and were transplanted, and none developed clinical evidence of posttransplant cholangiopathy., Conclusions: Biliary bicarbonate, pH, and glucose during ex situ NMP of liver grafts are accurate biomarkers of BDI and can be easily determined point of care, making them suitable for the pretransplant assessment of bile duct viability. This may improve graft selection and decrease the risk of posttransplant cholangiopathy.

Dual hypothermic oxygenated machine perfusion (DHOPE) of the liver has been advocated as a method to reduce ischemia/reperfusion injury (IRI). This study aimed to determine whether DHOPE reduces IRI of the bile ducts in donation after circulatory death (DCD) liver transplantation. In a recently performed phase 1 trial, 10 DCD livers were preserved with DHOPE after static cold storage (SCS; www.trialregister.nl NTR4493). Bile duct biopsies were obtained at the end of SCS (before DHOPE; baseline) and after graft reperfusion in the recipient. Histological severity of biliary injury was graded according to an established semiquantitative grading system. Twenty liver transplantations using DCD livers not preserved with DHOPE served as controls. Baseline characteristics and the degree of bile duct injury at baseline (end of SCS) were similar between both groups. In controls, the degree of stroma necrosis (P = 0.002) and injury of the deep peribiliary glands (PBG; P = 0.02) increased after reperfusion compared with baseline. In contrast, in DHOPE-preserved livers, the degree of bile duct injury did not increase after reperfusion. Moreover, there was less injury of deep PBG (P = 0.04) after reperfusion in the DHOPE group compared with controls. In conclusion, this study suggests that DHOPE reduces IRI of bile ducts after DCD liver transplantation. Liver Transplantation 24 655-664 2018 AASLD., (© 2018 The Authors Liver Transplantation published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Normothermic machine perfusion (NMP) enables viability assessment of donor livers prior to transplantation. NMP is frequently performed by using human blood products including red blood cells (RBCs) and fresh frozen plasma (FFP). Our aim was to examine the efficacy of a novel machine perfusion solution based on polymerized bovine hemoglobin-based oxygen carrier (HBOC)-201. Twenty-four livers declined for transplantation were transported by using static cold storage. Upon arrival, livers underwent NMP for 6 hours using pressure-controlled portal and arterial perfusion. A total of 12 livers were perfused using a solution based on RBCs and FFPs (historical cohort), 6 livers with HBOC-201 and FFPs, and another 6 livers with HBOC-201 and gelofusine, a gelatin-based colloid solution. Compared with RBC + FFP perfused livers, livers perfused with HBOC-201 had significantly higher hepatic adenosine triphosphate content, cumulative bile production, and portal and arterial flows. Biliary secretion of bicarbonate, bilirubin, bile salts, and phospholipids was similar in all 3 groups. The alanine aminotransferase concentration in perfusate was lower in the HBOC-201-perfused groups. In conclusion, NMP of human donor livers can be performed effectively using HBOC-201 and gelofusine, eliminating the need for human blood products. Perfusing livers with HBOC-201 is at least similar to perfusion with RBCs and FFP. Some of the biomarkers of liver function and injury even suggest a possible superiority of an HBOC-201-based perfusion solution and opens a perspective for further optimization of machine perfusion techniques. Liver Transplantation 24 528-538 2018 AASLD., (© 2017 The Authors. Liver Transplantation published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

In the majority of long-term survivors after PLTx, graft fibrosis has been identified. Recently, subtypes of graft fibrosis have been described based on their predominant acinar localization. We aimed to evaluate whether the development of portal, perisinusoidal, and centrilobular distribution of graft fibrosis is related to patient or transplantation-related parameters. We reviewed the histological features in protocol liver biopsies taken at 1 and 5 years after PLTx of 47 children on a tacrolimus-based immunosuppressive regimen. Fibrosis was assessed according to the LAFSc. The prevalence of portal fibrosis increased from 31% to 62%, sinusoidal from 68% to 79%, and centrilobular from 76% to 85%. The presence of portal fibrosis was associated with total bilirubin and γGT levels (each P<.02) and tended to be associated with biliary complications (P=.06). Sinusoidal fibrosis was associated with prior rejection episodes (P<.02) and centrilobular fibrosis with the presence of HLA mismatches (P=.02). In conclusion, using the LAFSc, we found a high incidence of progressive fibrosis in the 1-year and 5-year protocol biopsies after PLTx. Progression of fibrosis was observed in all acinar compartments, and each of the three locations is associated with different clinical conditions., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Lack of oxygen and biomechanical stimulation during static cold storage (SCS) of donor livers compromises endothelial cell function. We investigated the effect of end-ischemic oxygenated hypothermic machine perfusion (HMP) on endothelial cell function of extended criteria donor (ECD) livers., Methods: Eighteen livers, declined for transplantation, were transported to our center using static cold storage (SCS). After SCS, 6 livers underwent two hours of HMP, and subsequent normothermic machine perfusion (NMP) to assess viability. Twelve control livers underwent NMP immediately after SCS. mRNA expression of transcription factor Krüppel-like-factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) was quantified by RT-PCR. Endothelial cell function and injury were assessed by nitric oxide (NO) production and release of TM into the perfusate., Results: In HMP livers, mRNA expression of KLF2 (p = 0.043), eNOS (p = 0.028), and TM (p = 0.028) increased significantly during NMP. In parallel, NO levels increased during NMP in HMP livers but not in controls. At the end of NMP cumulative TM release was significantly lower HMP livers, compared to controls (p = 0.028)., Conclusion: A short period of two hours oxygenated HMP restores endothelial cell viability after SCS and subsequent normothermic reoxygenation of ECD livers., (Copyright © 2017 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Histologic scoring systems specific for primary sclerosing cholangitis (PSC) are not validated. We recently determined the applicability and prognostic value of three histological scoring systems in a single PSC cohort. The aim of this study was to validate their prognostic use and reproducibility across a multicenter PSC cohort. Liver biopsies from PSC patients were collected from seven European institutions. Histologic scoring was performed using the Nakanuma, Ishak, and Ludwig scoring systems. Biopsies were independently scored by six liver pathologists for interobserver agreement. The prognostic value of clinical, biochemical, and all three histologic scoring systems on predicting composite endpoints 1 (PSC-related death and liver transplantation), 2 (liver transplantation), and 3 (liver-related events), was assessed using univariable and multivariable Cox proportional hazards modeling. A total of 119 PSC patients were identified, and the median follow-up was 142 months. During follow-up, 31 patients died (20 PSC-related deaths), 31 patients underwent liver transplantation, and 35 patients experienced one or more liver-related events. All three staging systems were independent predictors of endpoints 2 and 3 (Nakanuma system: hazard ratio [HR], 3.16 [95% confidence interval (CI), 1.49-6.68] for endpoint 2 and HR, 2.05 [95% CI, 1.17-3.57] for endpoint 3; Ishak system: HR, 1.55 [95% CI, 1.10-2.18] for endpoint 2 and HR, 1.43 [95% CI, 1.10-1.85] for endpoint 3; Ludwig system: HR, 2.62 [95% CI, 1.19-5.80] for endpoint 2 and HR, 2.06 [95% CI, 1.09-3.89] for endpoint 3). Only the Nakanuma staging system was independently associated with endpoint 1: HR, 2.14 (95% CI, 1.22-3.77). Interobserver agreement was moderate for Nakanuma stage (κ = 0.56) and substantial for Nakanuma component fibrosis (κ = 0.67), Ishak stage (κ = 0.64), and Ludwig stage (κ = 0.62)., Conclusion: We confirm the independent prognostic value and demonstrate for the first time the reproducibility of staging disease progression in PSC using the Nakanuma, Ishak, and Ludwig staging systems. The Nakanuma staging system-incorporating features of chronic biliary disease-again showed the strongest predictive value. (Hepatology 2017;65:907-919)., (© 2016 by the American Association for the Study of Liver Diseases.)

Background: The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS)., Methods: Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37 °C) machine perfusion (NMP) after traditional SCS (0 °C-4 °C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12 °C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS., Results: During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups., Conclusions: Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

A short period of oxygenated machine perfusion (MP) after static cold storage (SCS) may reduce biliary injury in donation after cardiac death (DCD) donor livers. However, the ideal perfusion temperature for protection of the bile ducts is unknown. In this study, the optimal perfusion temperature for protection of the bile ducts was assessed. DCD rat livers were preserved by SCS for 6 hours. Thereafter, 1 hour of oxygenated MP was performed using either hypothermic machine perfusion, subnormothermic machine perfusion, or with controlled oxygenated rewarming (COR) conditions. Subsequently, graft and bile duct viability were assessed during 2 hours of normothermic ex situ reperfusion. In the MP study groups, lower levels of transaminases, lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances were measured compared to SCS. In parallel, mitochondrial oxygen consumption and adenosine triphosphate (ATP) production were significantly higher in the MP groups. Biomarkers of biliary function, including bile production, biliary bicarbonate concentration, and pH, were significantly higher in the MP groups, whereas biomarkers of biliary epithelial injury (biliary gamma-glutamyltransferase [GGT] and LDH), were significantly lower in MP preserved livers. Histological analysis revealed less injury of large bile duct epithelium in the MP groups compared to SCS. In conclusion, compared to SCS, end-ischemic oxygenated MP of DCD livers provides better preservation of biliary epithelial function and morphology, independent of the temperature at which MP is performed. End-ischemic oxygenated MP could reduce biliary injury after DCD liver transplantation., (© 2015 American Association for the Study of Liver Diseases.)

Background: Livers with moderate (30-60%) macrovesicular steatosis have been associated with poor outcome after transplantation. Aim of this study was to examine the outcome after transplantation of livers with moderate macrovesicular steatosis when the cold ischemia time (CIT) is kept very short., Methods: Postoperative outcome of 19 recipients of a moderate steatotic liver were compared with a matched control group of 95 recipients of a nonsteatotic liver graft (1:5 ratio). We studied graft/patient survival rates, incidences of primary nonfunction, postoperative complications (classified according to the Clavien-Dindo classification), first-week postoperative hepatic injury serum markers (AST/ALT), and liver function tests (PT time/bilirubin/lactate). In addition, we studied reversal of graft steatosis in follow-up biopsies., Results: Median CIT in livers with moderate steatosis and in controls was below 8 h in both groups. Although short- and long-term patient/graft survival rates and results of liver function tests were similar, serum markers of hepatic injury and postoperative complications (especially grade IVa) were significantly higher in recipients of a moderate steatotic liver. Reversal of steatosis was seen in 9 of the 11 (82%) recipients with follow-up liver biopsies., Conclusion: Despite the association with severe postoperative complications, moderate macrovesicular steatotic livers can be used successfully for transplantation if the CIT is kept very short., (© 2014 Steunstichting ESOT.)

Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37°C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial. [ABSTRACT FROM AUTHOR]